The present invention relates to an apparatus for isolating and purifying nucleic acids such as DNA and RNA or proteins such as enzymes and antibodies from biological materials such as microorganisms, e.g., viruses and bacteria, animal and plant tissues.
In purification of nucleic acids and proteins, an objective substance alone is separated from contaminants by various methods. Among these methods, generally conducted in the art are insolubilizing contaminants other than the objective substance and utilizing the difference in molecular weights between the objective substance and other contaminants in this technical field. Specifically, the separation of the objective substance from other contaminants is carried out by centrifugation, filtration, gel chromatography, etc.
The devices which automate these operations are commercially available, but they are not totally capable of fast processing of a plurality of samples. One of the reasons for this is that the devices need to be equipped with a centrifuge and filter attaching and detaching mechanism, thereby increasing the size of the devices and the number of steps in purification.
To solve the above problems, a method has been proposed to separate nucleic acids from contaminants using particles which can be magnetically drawn. The details of this method and the composition of reagents are described in Japanese Patent No. 2680462. The method does not require centrifugation or filtration and allows purification of nucleic acids in a single container. The kits for purifying nucleic acids and proteins utilizing the principle of the above patent have already been put on the commercial market (xe2x80x9cMagExtractorxe2x80x9d produced by Toyobo co., Ltd., etc.).
The purification of nucleic acids or proteins using magnetically attractable particles can be generally carried out by repeating the procedure comprising the following steps.
(a) A solution containing an objective substance and contaminants, a reagent and magnetically attractable particles are placed into a specific container, followed by thorough mixing (stirring). For this step, the technique by which only the objective substance is specifically bonded to magnetically attractable particles has been established.
(b) A magnet is placed close to the outside of the container to make the magnetically attractable particles gather at a position inside the container and separate the particles from a supernatant (solid-liquid separation). In the course of purification, this supernatant is discarded and only the objective substance, which is bonded to magnetically attractable particles, is left in the container. Generally, the steps of adding another reagent, mixing the mixture, solid-liquid separation of the mixture, removing the supernatant are repeated for a few times.
(c) To composite of the objective substance and the magnetically attractable particles is added a reagent which detaches only the objective substance from the magnetically attractable particles, and the magnet is placed close to the container again to make the magnetically attractable particles gather at a position inside the container. Hence, only the supernatant in the container is recovered in a different container, whereby the objective substance can be purified.
The apparatuses for purifying nucleic acids or proteins which automate the above operations (a), (b) and (c) are commercially available (xe2x80x9cMFX-2000xe2x80x9d manufactured by TOYOBO Co., LTD., etc.). This MFX-2000 comprises a piston pump and a nozzle capable of having a disposable tip which is attachable/detachable at the end. A common piston pump transfers water with a piston moving back and forth in a cylinder. In the field of this technique, a minute amount of a reagent is dispensed (distributed) and mixed (stirred) together with samples by discharging and sucking up the liquid according to the principle similar to that of this pump. In the actual operation, a disposable tip is attached to the end of the piston pump, and the liquid is sucked up into and discharged from inside the tip only (see FIG. 1). Such instrument is conventionally referred to as xe2x80x9cpipettemanxe2x80x9d. Further, the mixing or stirring by repeated sucking up a liquid into the tip and discharging the liquid using pipetteman is conventionally referred to as pipetting. Since pipetting is a piston action, it can be readily mechanized and applied to apparatuses for purifying nucleic acids or proteins.
In the above (a), mixing (stirring) is carried out by pipetting. Since the tip is disposable and attachable/detachable, it is convenient to avoid contamination between samples. In the above (b) and (c), solid-liquid separation is performed by discharging a liquid from inside the tip, stopping the piston pump, and placing the magnet close to the outside of the container. In this state, only the supernatant is sucked up. Thus, in the method for purifying nucleic acids or proteins using magnetically attractable particles, it is highly effective to use such mechanism equipped with a piston pump and a nozzle capable of having a disposable tip which is attachable/detachable at the end.
In this technical field, widely used are 96-hole plastic plates which comprises a series of holes in a grid pattern of 8 by 12 and are suitable for fast processing of a plurality of samples. Commercially available containers have the holes of a few xcexcl to a few ml capacity. The 96-hole plate advantageously allows simultaneous handling of 96 samples in a container and can be carried with one hand. In this technical field, fast processing of a plurality of samples is substantially impossible without using the devices for sampling, extraction, purification and analysis compatible with the format of this container (mainly the intervals between the holes in lengthwise and widthwise directions). In general, the containers having holes with some hundred xcexcl capacities are used for analysis. However, a container with holes of as great capacity as possible is ideal for extraction and purification in order to increase a recovery amount.
It is easily anticipated that the fast processing of a plurality of samples was enabled if the purification of nucleic acids or proteins using magnetically attractable particles can be performed using a 96 hole plate with the holes having as large capacity as possible. It is also considered that the automation of this processing could suitably use a mechanism comprising piston pumps equipped with nozzles capable of having disposable tips which are attachable/detachable at their ends. However, such automation entails the following problems.
I. Dispensing Method
As described in the above, the mixing (stirring) necessary for the purification of nucleic acids or proteins using magnetically attractable particles can be readily carried out by a mechanism comprising a piston pump and a nozzle capable of having a disposable tip which is attachable/detachable at the end. In conventional apparatuses, the operation of dispensing reagent necessary for purification is also conducted by a mechanism comprising a piston pump and a nozzle capable of having a disposable tip which is attachable/detachable at the end. This method is referred to as tip dispensing, and characteristically allows highly precise dispensing. In general, the purification of nucleic acids or proteins using magnetically attractable particles necessitates precise dispensing of reagents ranging from about several ten xcexcl to about several hundred xcexcl. For this reason, tip dispensing is considered suitable.
Reagents necessary for purification of nucleic acids or proteins using magnetically attractable particles are dispensed by tip dispensing comprising the following steps: placing a plurality of reagents necessary for purification in separate containers; moving a mechanism comprising a piston pump and a nozzle capable of having a disposable tip which is attachable/detachable at the end between a container, for example 96-hole plate used for purification and the reagent containers back and forth; and dispensing the reagents for a necessary number of times (repeating the cycle of suction of the reagents from the reagents container, moving, discharging the reagents into the container). Another possible mechanism consists of a fixed mechanism comprising a piston pump and a nozzle capable of having a disposable tip which is attachable/detachable at the end, wherein a container used for purification and a reagent container travel to the fixed mechanism. In both structures, it is time-consuming for the mechanism comprising a piston pump and a nozzle to travel between the container and the reagent containers used for purification, or for the container and the reagent containers used for purification to travel to the position of the mechanism comprising a piston pump and a nozzle. In addition, the space to travel back and forth unfavorably increases depending on the number of processing. This mechanism is also critically problematic in that the reagent drawn into the tips may drop during travelling and contaminate the samples. Moreover, because dispensing and mixing (stirring) can not be simultaneously performed, this system does not allow high-speed processing.
II. Wastes
When reagents necessary for purification are dispensed using a mechanism comprising a piston pump and a nozzle capable of having a disposable tip which is attachable/detachable at the end, tips have to be replaced and discarded for each reagent and, thereby increasing the amount of wastes. Thus, such dispensing is unfavorable. To achieve high-speed dispensing, such mechanisms are often equipped with multiple nozzles, which disadvantageously increases the number of tips to be used in proportion to the number of nozzles. Therefore, it is unfavorable.
III. Solid-liquid Separation
The shape of the magnet for drawing magnetically attractable particles contained in the respective holes of a 96-hole plate is illustrated in Japanese Patent No. 2701891. However, the 96-hole plate, each hole having a capacity of a few ml (2.2 ml), does not allow space for the magnet as in the method disclosed in Japanese Patent No. 2701891. Hence, it is impossible to efficiently perform solid-liquid separation with this place of the plate.
The purification of nucleic acids or proteins using magnetically attractable particles requires heating or cooling magnetically attractable particles in the plate before, after or during the solid-liquid separation. In conventional devices, the plate has to travel to a heating unit or cooling unit, or the samples have to be transferred to another plate which allows heating or cooling. Such constructions result in enlargement of the devices and inefficient purification because of the loss of the samples.
IV. Methods for Mixing (Stirring) and Dispensing Suspension Containing Magnetically Attractable Particles
Because magnetically attractable particles usually contain iron oxide and the like, they are very likely to precipitate in the suspension containing these particles. Japanese Unexamined Patent Publication No. 1998-316696 describes the method for lowering the precipitating rate by increasing the specific gravity of the liquid, and for facilitating the resuspension of precipitated particles. This method enables a mechanism equipped with a piston pump and a nozzle capable of having a disposable tip which is attachable/detachable at the end to resuspend (mix (stir)) magnetically attractable particles in a suspension containing the particles which are precipitated to a certain degree. However, some magnetically attractable particles can not be suspended again after being left for a few days by the above mechanism. Therefore, the particles need to be suspended manually before use. Hence, automation of suspending such magnetically attractable particles is difficult for a mechanism comprising a piston pump and a nozzle capable of having a disposable tip which is attachable/detachable at the end, especially in intermittent operations.
V. Full Automation
When nucleic acids or proteins are purified from microorganisms or cultured cells using 96-hole plates, in order to prepare pellets containing cells, a culture liquid in which microorganisms or cultured cells float should be preliminarily centrifuged using a specific centrifuge for a 96-hole plate, or a supernatant of the culture liquid should be removed using a filter before the purification step. Accordingly, automation of a series of steps from sampling to purification is difficult for a mechanism by itself comprising piston pumps and nozzles capable of having disposable tips which are automatically attachable/detachable. Stated more specifically, the automation of the steps requires a centrifuge, a vacuum pump for filtration and other devices.
VI. Contamination Prevention
In the method comprising sucking up a liquid into the tips and discharging the liquid, sucking up the whole amount can not be achieved without sucking a little air in addition to the actual amount of the sucked liquid (necessity of excess suction). Further, discharging the whole amount of the liquid is impossible without discharging a little air in addition to the whole amount of the sucked liquid and the sucked air. When the liquid to be sucked and discharged contains surfactants or like substances which are prone to foaming, the excess air sucked or discharged inevitably causes foaming at the tip ends. When a plurality of piston pumps and nozzles capable of having disposable tips which are attachable/detachable are arranged to suit the arrangement of the holes of a 96-hole plate, the distances in the lengthwise and widthwise directions between the tips are greatly narrowed. The foams are very likely to contact those of the adjacent tips, posing the problems of contamination between the tips and inaccuracy of experiments and analyses.
In view of the above problems, the present inventors have conducted extensive research. Consequently, the inventors found that the foregoing problems in an apparatus for purifying nucleic acids or proteins can be solved by providing a mechanism for mixing a liquid mixture of each sample and a reagent in a plurality of sections of a container by sucking up a plurality of the mixtures into tips, followed by discharging the mixtures in the sections simultaneously, and a mechanism for dispensing a desired amount of a reagent to be used subsequently into the same number of sections of another container as samples, while the mixing is in progress. The present invention was accomplished based on this finding.
The present invention relates to the following 1 to 15:
1. An apparatus for purifying nucleic acids or proteins comprising:
a plurality of piston pumps; and
a plurality of nozzles capable of having disposable tips which are automatically attachable/detachable,
the apparatus being characterized by comprising:
a mechanism for mixing a liquid mixture of each sample and a reagent in a plurality of sections of a container by sucking up a plurality of the mixtures into tips, followed by discharging the mixtures in the sections simultaneously; and
a mechanism for dispensing a desired amount of a reagent to be used subsequently into the same number of sections of a different container as samples, while the mixing is in progress.
2. An apparatus for purifying nucleic acids or proteins comprising:
96 piston pumps; and
96 nozzles capable of having 96 disposable tips which are automatically attachable/detachable
the apparatus being characterized by comprising:
a mechanism for mixing a liquid mixture of each sample and a reagent in a multiple of 96 of sections of a container by sucking up a plurality of the mixtures into tips, followed by discharging the mixtures in the sections simultaneously; and
a mechanism for dispensing a desired amount of a reagent to be used subsequently into the same number of sections of a different container having sections of a multiple of 96 as samples, while the mixing is in progress.
3. An apparatus for purifying nucleic acids or proteins, the apparatus comprising:
a mechanism for removing each supernatant from a suspension of magnetically attractable particles in a multiple of 96 of sections of a container using 96 hollow cylindrical magnets; and
a mechanism for heating or cooling the container through the magnets,
wherein the purification of nucleic acids or proteins uses magnetically attractable particles.
4. An apparatus for purifying nucleic acids or proteins, the apparatus comprising:
a drive mechanism for a plurality of piston pumps;
a mechanism for driving said the drive mechanism up and down; and
magnets, a heating device and a cooling device in the direction the drive mechanism is driven.
5. An apparatus for purifying nucleic acids or proteins, the apparatus comprising:
a drive mechanism for a plurality of piston pumps;
a mechanism for driving said drive mechanism up and down;
a plate comprising a plurality of magnets having the similar arrangement to that of piston pumps directly beneath said piston pumps;
a mechanism capable of heating and cooling under said plate; and
a mechanism for conveying a container containing samples between said piston pumps and said plate.
6. An apparatus for purifying nucleic acids or proteins comprising:
a plurality of piston pumps; and
a plurality of nozzles capable of having disposable tips which are automatically attachable/detachable,
the apparatus being characterized by comprising:
a mechanism for sucking up each liquid not containing the objective substance in a plurality of sections of a container and discharging the liquids in sections of a liquid waste container, the sections of the liquid waste container having the same arrangement according to the format of the plurality of piston pumps, using a plurality of piston pumps and a plurality of nozzles capable of having disposable tips which are automatically attachable/detachable.
7. A method for purifying nucleic acids or proteins using the apparatus for purifying nucleic acids or proteins according to any of items 1 to 6,
comprising sucking up each liquid not containing the objective substance into tips using a plurality of piston pumps and a plurality of nozzles capable of having disposable tips which are automatically attachable/detachable, and discharging the liquids from the tips into sections of a liquid waste container, the sections of which bottoms are inclined in a vertical cross-sectional view and being provided with holes of which diameter is smaller than the outer diameter of the tip end near the center of the bottom.
8. The method for purifying nucleic acids or proteins according to item 7, wherein magnetically attractable particles are used.
9. The method for purifying nucleic acids or proteins according to item 8, wherein the suspension of the magnetically attractable particles and a liquid are mixed by decreasing the pressure inside the container containing said suspension and discharging a certain amount of said suspension with the application of pressure greater than atmospheric pressure.
10. The apparatus for purifying nucleic acids or proteins according to any of items 1 to 6, the apparatus comprising a mechanism which is capable of conducting the method of item 9 using an air pump and an electromagnetic valve.
11. A method for purifying nucleic acids or proteins using the apparatus for purifying nucleic acids or proteins according to any of items 1 to 6, wherein a plurality of roller tube pumps are used for transferring and dispensing the reagents, each roller tube pump being connected to tubes having different diameters, and a reagent inlet and reagent outlet of each tube being common.
12. A method for purifying nucleic acids or proteins according to item 11, wherein dispensing is carried out alternately with normal rotation and reverse rotation of rollers.
13. The apparatus for purifying nucleic acids or proteins according to items 1 to 6, the apparatus comprising a mechanism which is capable of conducting the method according to item 11 or 12.
14. A method for purifying nucleic acids using magnetically attractable particles, the method comprising the steps of:
adding magnetically attractable particles to a cell culture solution or suspension to collect the cells;
adding a reagent for dissolving the cells to make contaminants other than an objective nucleic acid adsorb onto said particles;
separating a supernatant from said particles; and
adding another magnetically attractable particles to the supernatant.
15. The apparatus for purifying nucleic acids or proteins according to any of items 1 to 6, wherein the apparatus comprise a software which is capable of conducting the method of item 14.
In the present specification, by the term xe2x80x9cliquidxe2x80x9d is meant not only a liquid, but also a suspension.